During the last 5 years, PI was able to utilize the current K05 award to develop two new NIDA funded projects in his laboratory, and was able to spend a semester leave in Dr. Chris Evans'laboratory at UCLA to explore the use zebrafish as an alternative model for studying the molecular mechanism of opiate tolerance and dependence, a life-long career goal of PI. This was made possible because of the relief from his administrative and teaching commitments at University of Minnesota due to the K05 award. Therefore, the objective of this K05 award application remains to be a mechanism allowing PI to continue his successful program of taking periodic leaves of absence from University of Minnesota and spend time in his collaborator's laboratory in the pursuit of new or alternative approaches and technologies in studying the molecular mechanism of tolerance and dependence. It is clear from the on-going projects in PI's laboratory that opioid receptor signals via the formation of receptorsomes. The scaffolding of cellular proteins with opioid receptor within the microdomains greatly affect the receptor signaling. By recruiting different proteins to the receptor vicinity, e.g., beta-arrestin, Src, RGS and AGS, the magnitude and duration of signals could be modulated. PI has initiated studies to identify the cellular proteins that could modulate opioid receptor activities. Using yeast two-hybrid screens of mouse brain library, protein candidates, such as the FK506 binding protein FKBP12 that specifically interacts with the carboxyl tail domain of MOR could modulate the agonist-induced intracellular Ca2+ movement. However, the use of a specific receptor domain limits the identification of proteins that require multiple domains or tertiary receptor structure for binding. Thus we will continue our on-going projects to determine the components of opioid receptorsomes by the use of proteomic approaches and yeast two-hybrid screens using whole receptor protein. Over-expression of these proteins with adenoviruses, or the knockdown of these proteins levels in neuroblastoma N2A cells by siRNA will be carried out to determine their effects on two the effectors regulated by opioid receptors, i.e., adenylyl cyclase and intracellular Ca2+ homeostasis. The alteration of these proteins levels in primary hippocampal cultures enriched in neurons expressing endogenous MOR will be carried out also. The inducible siRNA vector will be developed and used in the temporal knockdown of the proteins involved in the receptorsomes formation. Genetic alteration of the proteins levels will be carried out in mice and other models to test the effects of these proteins in chronic opiate drug actions.